N-thiolated 2-oxazolidinone -derived antibiotics

ABSTRACT

This invention describes the discovery and synthesis of N-thiolated 2-oxazolidinones as a new class of anti bacterial agents. These compounds can be synthesized from 2-oxazolidinones by Ndeprotection and N-sulfenylation. These new substances were found to exhibit potent anti-bacterial activity, including bacteriostatic properties against  Staphylococcus  spp., including methicillin resistant  Staphylococcus aureus  (MRSA), and  Bacillus  spp., including  Bacillus anthracis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending U.S. Nonprovisional patentapplication Ser. No. 11/382,157, entitled “N-Thiolated 2-OxazolidinoneAntibiotics”, filed May 8, 2006, which claims priority to U.S.Provisional Patent Application 60/678,292, entitled, “N-Thiolated2-Oxazolidinones: A New Class of Anti-bacterial Drug”, filed May 6,2005, the contents of which are herein incorporated by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Grant No. R01 AI51351 awarded by the National Institutes of Health. The Government hascertain rights in the invention.

FIELD OF INVENTION

This invention pertains to the synthesis and characterization of a newfamily of antibacterial drug. More specifically this invention relatesto N-thiolated 2-oxazolidinones as a new class of syntheticantibacterial agents.

BACKGROUND OF THE INVENTION

The clinical use of antibiotics in the 20th century has substantiallydecreased morbidity from bacterial infections. The early success ofpenicillin was extended by various sulfonamide drugs developed in the1930s, and subsequently by a “golden” period of discovery, between 1945and 1970, during which a wide array of highly effective agents arediscovered and developed (Chopra, I., et al., “The Search forAntimicrobial Agents Effective against Bacteria Resistant to MultipleAntibiotics” Antimicrobial Agents and Chemotherapy, 1997, 41:497-503).

However, since the 1980s the introduction of new antibiotics has slowed,and, concurrently, there has been an alarming increase in bacterialresistance to existing agents that now constitutes a serious threat topublic health (Brown, A. G. “Discovery and Development of New β-LactamAntibiotics” Pure & Appl. Chem., 1987, 59:475-484). Hospitals, nursinghomes and infant day care centers have become breeding grounds for themost tenacious drug-resistant pathogens (“Frontiers in Biotechnology”Science, 1994, 264:359-393). There has been an alarming rise in drugresistant staphylococci, enterococci, streptococci, and pneumococciinfections, and a rise in tuberculosis, influenza and sepsis.

The problem of bacterial drug resistance has reached a crisis level suchthat successful treatment of antibiotic-resistant infections inhospitals and health care centers can no longer be taken for granted.Infections caused by methicillin-resistant Staphylococcus aureus (MRSA)are becoming particularly difficult to treat with conventionalantibiotics such as penicillin, leading to a sharp rise in clinicalcomplications and deaths. The need for new antibacterial agents andprotocols for treating MRSA infections is becoming extremely serious.

A novel family of lipophilic N-thiolated β-lactams that are effectivegrowth inhibitors of MRSA and Bacillus species has been reported (U.S.Pat. No. 6,473,015 B1 to Turos et al. and U.S. Pat. No. 6,946,458 B2 toTuros; see also. Ren, X. F. et al., J. Org. Chem. 60 (1995), p. 4980;Ren, X. F. et al., J. Org. Chem. 63 (1998), p. 8898; E. Turos, E. etal., Tetrahedron 56 (2000), p. 5571; E. Turos, E. et al., Bioorg. Med.Chem. Lett. 12 (2002), p. 2229; C. Coates, C. et al., Bioorg. Med. Chem.11 (2003), p. 193; Long, E. et al., Bioorg. Med. Chem. 11 (2003), p.1859; Kazi, A. et al., Biochem. Pharmacol. 67 (2004), p. 365; Turos, E.et al., J. Bioorg. Med. Chem. Lett. 2006 (in press)). The mode of actionand structure-activity profiles differ dramatically from those oftraditional β-lactams. (See generally Chemistry and Biology of β-LactamAntibiotics; Morin, R. B., Gorman, M.; Eds.; Academic Press: New York,1982; Vols 1-3.) Investigations have shown that these β-lactam compoundscan carry a wide range of substituents at the C₃ and C₄ centers;however, the N-organothio substituent is necessary for microbiologicalactivity. (E. Turos, E. et al., Bioorg. Med. Chem. 13 (2005), p. 6289.)The mechanism of action is under investigation but appears to depend onthe ability of the compounds to transfer the organothio moiety onto acellular thiol. This suggests that the role of the lactam ring is toprovide a structural framework for the delivery of the thiol moiety andmay not be absolutely required for the activity. To probe thispossibility, and to expand on the structural diversity of anti-MRSAcompounds available for clinical development, oxazolidinones wereexamined as potential antibacterially active organothio carriers.Oxazolidinones are already recognized for their favorablepharmacological properties and are the only new class of antibacterialdrugs introduced into clinical use in the last three decades. (Brickner,S., J. Curr. Pharm. Des. 2 (1996), p. 175; Phillips, O. A., Curr. Opin.Invest. Drugs 4 (2003), p. 117; S. J. Brickner, S. J. et al., J. Med.Chem. 39 (1996), p. 673.)

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA)are becoming extremely difficult to treat with conventional antibiotics,leading to a sharp rise in clinical complications (Binder, S. et al.Science, 1999, 284:1311). The need for new antibiotics and protocols fortreating MRSA infections is extremely serious.

There is a clear need for new antibacterial agents to combat pathogenicbacteria that have become resistant to current antibiotics. Towards thisend, a novel class of derivatized, N-thiolated-2-Oxazolidinones havebeen developed in the present invention, that exhibit strongantibacterial activity against a wide variety of species and strains,including methicillin-resistant Staphylococcus aureus.

SUMMARY OF INVENTION

This invention pertains to the synthesis of a new family ofantibacterial drug. N-thiolated-2-Oxazolidinones represent a new classof antibacterial agent for methicillin-resistant Staphylococcus aureus.Described herein is the synthesis and application of N-thiolated2-oxazolidinones as a new class of anti bacterial agents. Thesecompounds can be synthesized from 2-oxazolidinones by N-deprotection andN-sulfenylation. These new substances were found to exhibit potentanti-bacterial activity, including bacteriostatic properties againstmethicillin resistant Staphylococcus aureus (MRSA).

The general structure of these N-thiolated 2-oxazolidinones is:

wherein R₁₋₅ are independently hydrogen, alkyl, heteroalkyl, aryl,heteroaryl, alkenyl, or alkynyl; X is H, C or O; and n=0 to 3.

It is an object of the present invention to provide these compounds,including their salts, hydrates, and in combinations with suitablepharmaceutical carriers, as antibacterial and antibiotic agents.

It is a further object of this invention to provide such compounds,wherein R₄ and R₅ are hydrogen, and —C(R₁)₃ is aryl or heteroaryl.

It is a further object of this invention to provide antibacterial andantibiotic agents with varying bacterial strain specificities andefficacies, by the expedient means of varying substituents of the2-oxazolidinone ring, including but not limited to nitrogen (N-1)methylthio or benzylthio moieties, and substitutions at the C₃ and C₄positions.

In certain embodiments the present invention provides methods forinhibiting the growth of bacteria by administering the compounds of thepresent invention, and to provide methods for the treatment of bacterialinfections of a patient, in which one or more doses of an effectiveamount of the compounds and compositions of the present invention areadministered to a patient.

The present invention provides a method of inhibiting a bacterialinfection comprising administering an effective amount of theN-thiolated 2-oxazolidinone of claim 1, to a patient in need thereof.

In certain embodiments the bacterium is a Staphylococcus spp. In certainspecific embodiments the Staphylococcus spp. is a methicillin-resistantStaphylococcus. In still further embodiments the methicillin-resistantStaphylococcus can be MRSA USF919, MRSA USF920, MRSA USF652, MRSAUSF653, MRSA USF654, MRSA USF655, MRSA USF656, MRSA USF657, MRSA USF658or MRSA USF659.

In certain embodiments the bacterium is a Bacillus spp. In still furtherembodiments the Bacillus spp. can be B. anthracis, B. globigii, B.thurigensis, B. megaterium, B. subtilis, B. cereus and B. coagulans.

In certain embodiments the present invention provides compounds andcompositions suitable for the treatment of Staphylococcus spp.infection.

In certain embodiments the present invention provides a method ofinhibiting Staphylococcus spp. infection. In further embodiments thepresent invention provides a method of inhibiting methicillin-resistantStaphylococcus aureus infection.

In certain embodiments the present invention provides compounds andcompositions suitable for the treatment of Bacillus spp. infection.

In certain embodiments the present invention provides a method ofinhibiting Bacillus spp. infection. In further embodiments the presentinvention provides a method of inhibiting methicillin-resistantStaphylococcus aureus infection.

It is a further object of this invention to provide a mechanism ofinhibiting infection comprising administering an N-thiolated2-oxazolidinone antibacterial compound to a patient in need thereof,where said antibacterial compound affects events within the cytoplasm ofthe cell.

It is a further object of this invention to provide a mechanism ofinhibiting bacterial infection by a means other than inhibiting cellwall cross-linking.

It is a further object of this invention to provide a mechanism ofinhibiting bacterial infection through the use of an antibacterialcompound that does not block bacterial cell growth by inhibitingpenicillin binding proteins.

The present invention confers numerous advantages over the compounds ofthe prior art, including the following: ease of synthesis, wherebycompounds with diverse substitutents may be synthesized and tested forantibacterial and antibiotic activity; the invention provides novelantibacterial and antibiotic agents to which bacterial pathogens havenot yet acquired resistance; and the invention provides novel compoundsfor the treatment of increasingly common and resistant diseases.Surprisingly, the inventors have found that antibacterial and antibioticactivities can be obtained in compounds that do not possess traditionalactivating groups attached to the nitrogen, as required for activity inconventional monobactams which contain, for example, a sulfonic acidgroup. The inventors have also surprisingly discovered thatderivatization of structure (A) in FIG. 1 at the positions indicated bythe R₁₋₅ and X, results in compounds exhibiting different specificitiesfor different bacterial pathogens, in a manner that is currently notpossible to predict a priori. This aspect is therefore an unobviousbenefit of the present invention. The present invention fulfills anurgent need in that novel compounds are urgently required as bacterialpathogens increasingly acquire immunity towards the present arsenal ofantibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 shows an N-methylthio 2-oxazolidinone.

FIG. 2 shows an N-thiolated β-lactam.

FIG. 3 shows a reference N-thiolated β-lactam (Lac) and N-alkylthio2-oxazolidinones 1-5.

FIG. 4 shows enantiomerically pure N-methylthio 2-oxazolidinones 6-9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention pertains to the synthesis of a new family ofantibacterial drug. N-thiolated-2-Oxazolidinones represent a new classof antibacterial agent for methicillin-resistant Staphylococcus aureus.Described herein is the synthesis and application of N-thiolated2-oxazolidinones as a new class of anti bacterial agents. Thesecompounds can be synthesized from 2-oxazolidinones by N-deprotection andN-sulfenylation. These new substances were found to exhibit potentanti-bacterial activity, including bacteriostatic properties againstmethicillin resistant Staphylococcus aureus (MRSA).

The term “N-thiolated-2-Oxazolidinones” is used herein to refer to acyclic 5-membered compound comprising a 2-Oxazolidinone ring in whichthe ring nitrogen (N1) atom is covalently bonded to a sulfur that iscovalently bonded to a carbon-centered moiety, and which may be furthermodified as described herein. Specifically, referring now to compound(A) as shown in FIG. 1, X may be a hydrogen (in which case, n ispreferably zero), or a carbon atom (in which case, n is preferably 3),or an oxygen atom (in which case, n is preferably 1), and R₂ may be anysubstituent as herein defined. Similarly, R₁ and R₃₋₅ may beindependently any substituent as herein defined.

Thus, in advantageous embodiments, R₁ is hydrogen or benzyl, and inparticularly advantageous embodiments R₁ is hydrogen. Substituentscomprising —X(R₂)_(n) are preferably methoxy and hydrogen, and mostpreferably methoxy. R₃ may be alkyl, heteroalkyl, aryl, heteroaryl,alkenyl, or alkynyl. Preferred R₃ substituents are phenylethynyl,acetoxy, 1-propenyl, ortho-chlorophenyl, ortho-nitrophenyl, 2-thiophene,or S,S-dioxo-thiophene. R₄ and R₅ may be independently alkyl,heteroalkyl, aryl, heteroaryl, alkenyl, or alkynyl groups. In preferredembodiments, R₄ and R₅ are H.

The following definitions are used, unless otherwise described. Halo isfluoro, chloro, bromo, or iodo. “Alkyl,” “alkoxy,” etc. denote bothstraight and branched groups; but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical, a branchedchain isomer such as “isopropyl” being specifically referred to. “Aryl”denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radicalhaving about nine to ten ring atoms in which at least one ring isaromatic. “Heteroaryl” encompasses a radical attached via a ring carbonof a monocyclic aromatic ring containing five or six ring atomsconsisting of carbon and one to four heteroatoms each selected from thegroup consisting of non-peroxide oxygen, sulfur, and N(R_(x)) whereinR_(x) is absent or is hydrogen, oxo, alkyl, phenyl or benzyl, as well asa radical of an ortho-fused bicyclic heterocycle of about eight to tenring atoms derived therefrom, particularly a benz-derivative or onederived by fusing a propylene, trimethylene, or tetramethylene diradicalthereto. “Heteroalkyl” encompasses the replacement of a carbon atomwithin an alkyl chain with a heteroatom; e.g., replacement with anelement other than carbon such as N, S, or O, including both an alkylinterrupted by a heteroatom as well as an alkyl substituted by aheteroatom.

It will be appreciated by those skilled in the art that compounds of theinvention having one or more chiral center(s) may exist in and beisolated in optically active and racemic forms. Some compounds mayexhibit polymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis, from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase), and how to determine antibacterial activity using thetests described herein, or using other tests which are well known in theart.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

Specifically, “alkyl” can include methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl,nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl or pentadecyl;“alkenyl” can include vinyl, 1-propenyl, 2-propenyl, 1-butenyl,2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl,2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 1-nonenyl, 2-nonenyl,3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl,1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl,7-decenyl, 8-decenyl, 9-decenyl; 1-undecenyl, 2-undecenyl, 3-undecenyl,4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl,9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl,4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl,9-dodecenyl, 10-dodecenyl, 11-dodecenyl, 1-tridecenyl, 2-tridecenyl,3-tridecenyl, 4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl,8-tridecenyl, 9-tridecenyl, 10-tridecenyl, 11-tridecenyl, 12-tridecenyl,1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl, 4-tetradecenyl,5-tetradecenyl, 6-tetradecenyl, 7-tetradecenyl, 8-tetradecenyl,9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl, 12-tetradecenyl,13-tetradeceny, 1-pentadecenyl, 2-pentadecenyl, 3-pentadecenyl,4-pentadecenyl, 5-pentadecenyl, 6-pentadecenyl, 7-pentadecenyl,8-pentadecenyl, 9-pentadecenyl, 10-pentadecenyl, 11-pentadecenyl,12-pentadecenyl, 13-pentadecenyl, 14-pentadecenyl; “alkoxy” can includemethoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy,pentoxy, 3-pentoxy, hexoxy, heptyloxy, octyloxy, nonyloxy, decyloxy,undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, or pentadecyloxy;“alkanoyl” can include acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl,heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl,tridecanoyl, tetradecanoyl, or pentadecanoyl; “cycloalkyl” can includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl. “Aryl” can include phenyl, indenyl,5,6,7,8-tetrahydronaphthyl, or naphthyl. “Heteroaryl” can include furyl,imidazolyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl(or its N-oxide), indolyl, or quinolyl (or its N-oxide).

Specific independent values for R₁₋₅, include alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkyl(C₁-C₁₀)alkyl, (C₃-C₈)cycloalkyl(C₁-C₁₅)alkenyl,(C₃-C₈)cycloalkyl(C₁-C₁₅)alkynyl, (C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl, or(C₁-C₁₅)alkanoyloxy; wherein R¹ is optionally substituted with one ormore (e.g., 1, 2, 3, or 4) substituents independently selected from thegroup consisting of halo, nitro, cyano, hydroxy, trifluoromethyl,trifluoromethoxy, (C₁-C₁₅)alkyl, (C₂-C₁₅)alkenyl, (C₂-C₁₅)alkynyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl, (C₁-C₁₅)alkyl,(C₃-C₈)cycloalkyl-(C₂-C₁₅)alkenyl, (C₃-C₈)cycloalkyl(C₂-C₁₅)alkynyl,(C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl, (C₁-C₁₅)alkanoyloxy, C(═O)OR_(a),C(═O)NR_(b)R_(c), OC(═O)OR_(a), OC(═O)NR_(b)R_(c), AND NR_(e)R_(f).

Other specific values for R₁₋₅ include aryl optionally substituted with1, 2, or 3 substituents independently selected from the group consistingof halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy,(C₁-C₆)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl, (C₁-C₆)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkanoyl,(C₂-C₁₀)alkanoyloxy, C(═O)OR_(a), C(═O)NR_(b)R_(c), or NR_(e)R_(f).

Other specific values for R₁₋₅, include independently phenyl ornaphthyl, optionally substituted with a substituent selected from thegroup consisting of halo, nitro, cyano, hydroxy, trifluoromethyl,trifluoromethoxy, (C₁-C₆)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₁-C₁₀)alkoxy,(C₁-C₁₀)alkanoyl, (C₂-C₁₀)alkanoyloxy, C(═O)OR_(a), C(═O)NR_(b)R_(c), orNR_(e)R_(f).

Still other specific values for R₁₋₅, include aryl, heteroaryl,aryl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl, aryl(C₂-C₆)alkenyl,heteroaryl(C₂-C₆)alkenyl, aryl(C₂-C₆)alkynyl, orheteroaryl(C₂-C₆)alkynyl; wherein any aryl or heteroaryl is optionallysubstituted with halo, nitro, cyano, hydroxy, trifluoromethyl,trifluoromethoxy, (C₁-C₁₅)alkyl, (C₂-C₁₅)alkenyl, (C₂-C₁₅)alkynyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₁₅)alkyl,(C₃-C₈)cycloalkyl-(C₂-C₁₅)alkenyl, (C₃-C₈)cycloalkyl(C₂-C₁₅)alkynyl,(C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl, (C₁-C₁₅)alkanoyloxy, C(═O)OR_(a),C(═O)NR_(b)R_(c), or NR_(e)R_(f).

The compounds of the present invention exhibit broad antibacterialactivity against several families of bacteria in the Gram-negative andGram-positive range, and against beta-lactamase formers. Because oftheir powerful antibacterial properties, the present compounds may alsobe used to supplement feed for animals.

In addition, the compounds of the present invention that exhibitantibacterial activity may also be used as medicaments, and also assubstances for preserving inorganic and organic materials, especiallyorganic materials of all kinds, for example, polymers, lubricants,paints, fibers, leather, paper, timber, foodstuffs, and water. Forexample, these compounds can be covalently bonded to the polymer.

The compounds of the present invention may also be used to prevent,alleviate, or cure diseases caused by pathogens whose growth isinhibited by these compounds. The instant compounds are particularlyactive against bacteria and bacteria-like microorganisms. They aretherefore suitable for use in human and veterinary medicine, for theprophylaxis and chemotherapy of local and systemic infections caused bythese pathogens.

As an illustrative, but not limiting, list of pathogens, the followingpathogenic microorganisms are possible targets of the compounds of thepresent invention. Micrococcaceae, such as Staphylococci, for exampleStaphylococcus aureus, Staph. Epidermidis and Staph. Aerogenes;Lactobacteriaceae, such as Streptococci, for example Streptococcuspyogenes; Neisseriaceae, such as Neisseriae, for example Neisseriagonorrhoeae (Gonococci); Corynebacteriaceae, such as Corynebacteria;Listeria bacteria; Erysipelothrix bacteria; Kurthia bacteria;Enterobacteriaceae, such as Escherichia bacteria of the Coli group;Klebsiella bacteria; Erwiniae; Serratia; Proteae bacteria; Providenciabacteria; Salmonella bacteria; Shigella; Pseudomonadaceae; Aeromonasbacteria; Spirillaceae, such as Vibrio bacteria; Spirillum bacteria;Parvobacteriaseae; Brucella bacteria; Bordetella bacteria; Moraxellabacteria; Fusiform bacteria; Bacillaceae; Clostridia; Spirochaetaceae;Treponema bacteria; and Leptospira bacteria.

Examples which may be cited of diseases which can be prevented,alleviated, or cured by the compounds of the present invention are:diseases of the respiratory passages and of the pharyngeal cavity;otitis; pharyngitis; pneumonia; peritonitis; pyelonephritis; cystitis;endocarditis; systemic infections; and bronchitis.

The compounds of the present invention include all hydrates and saltsthat can be prepared by those of skill in the art. Under conditionswhere the compounds of the present invention are sufficiently basic oracidic to form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids whichform a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a patient, such as ahuman patient, in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical, or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water or other suitablesolvent, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredientpresenting the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied inpure-form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from adsorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of formula I to the skin aredisclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art (U.S. Pat. No.4,938,949 (Borch et al.)).

Generally, the concentration of the compound(s) of formula (I) in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

Accordingly, the invention includes a pharmaceutical compositioncomprising a compound of the present invention as described above; or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofone or more compounds effective to treat a bacterial infection, are apreferred embodiment of the invention.

The present invention provides a novel class of monocyclic substituted2-oxazolidinones, specifically termed N-thiolated 2-oxazolidinones asdefined herein.

The present invention will therefore be fully understood by one of skillin the art by reference to the following embodiments, examples, andclaims.

Example 1

In accordance with the present invention, a representative selection ofdifferentially substituted N-thiolated 2-oxazolidinones 1-9 was preparedfor antimicrobial screening by N-thiolation of the corresponding2-oxazolidinones using protocols that have been previously reported byMiller and co-workers (Woulfe, S. R. et al., Tetrahedron Lett. 1985, 26,3891) for β-lactams. The structures of the compounds were confirmed by¹H and ¹³C NMR spectroscopy, and antibacterial assays were performed byKirby-Bauer disk diffusion on agar plates according to NCCLS guidelines.(NCCLS (National Committee for Clinical Laboratory Standards) Methodsfor Dilution of Antimicrobial Susceptibility Tests for Bacteria thatGrow Aerobically. NCCLS Document M7-A4, Vol. 17, No. 2, 1997) Theseassays tested both an ATCC strain of methicillin-susceptible S. aureusas well as 10 strains of methicillin-resistant S. aureus obtained eitherfrom ATCC sources or as clinical isolates from a local hospital. Thezones of growth inhibition produced by the compounds against each ofthese microbes after 24 h of incubation are presented in Table 1.Compound susceptibility measurements obtained from agar disk diffusionof oxazolidinones 1-9 against a methicillin-susceptible strain ofStaphylococcus aureus (S. aureus ATCC 25923) and 10 strains ofmethicillin-resistant S. aureus

TABLE 1 (MRSA) Bacterial strains Lac 1 2 3 4 5 6 7 8 9 Pen G S. aureus849 (ATCC 25923) 25 31 16 17 21 19 30 29 20 21 33 MRSA USF919 (ATCC43300) — 32 11 19 20 19 30 28 21 13 — MRSA USF920 (ATCC 33591) — 32 0 1916 19 28 28 18 15 — MRSA USF652 30 30 12 17 16 19 30 30 19 12 8 MRSAUSF653 30 32 19 20 20 20 31 30 27 25 15 MRSA USF654 26 30 14 18 20 22 2926 23 20 10 MRSA USF655 25 30 14 17 20 22 29 28 22 18 14 MRSA USF656 2831 15 19 22 20 31 28 22 20 12 MRSA USF657 27 30 12 19 21 22 29 27 22 1912 MRSA USF658 26 31 14 17 18 21 29 27 16 19 19 MRSA USF659 24 28 16 1919 20 26 26 22 22 16

The first goal was to determine the effect of substitution at the C₄ andC₅ centers of the oxazolidinone ring on anti-Staphylococcus activity.Accordingly, N-thiolated oxazolidinones 1-5 (as racemates) were examinedand compared to two reference compounds, N-methylthio lactam Lac (FIG.2) and penicillin G.

Compound susceptibility measurements obtained from agar disk diffusionof oxazolidinones 1-9 against a methicillin-susceptible strain ofStaphylococcus aureus (S. aureus ATCC 25923) and 10 strains ofmethicillin-resistant S. aureus (MRSA) In each case, 20 μg of the testcompound in CH₂Cl₂ was applied to 6 mm cellulose disks prior toinoculation and incubation. The value corresponds to average diameter inmm (triplicate experiments) for the zone of growth inhibitions observedafter 24 h of incubation at 37° C. S. aureus (ATCC 25923) andmethicillin-resistant S. aureus (labeled MRSA USF652-659 and USF919-920)were obtained from Lakeland Regional Medical Center, Lakeland, Fla. Lacis the N-thiolated β-lactam shown in FIG. 2. Pen G is penicillin G(potassium salt). Error values are within ±1 mm.

In almost every case, the five oxazolidinones displayed about equalactivity against both S. aureus and MRSA, as did the correspondingβ-lactam (Lac), and were uniformly much more effective than penicillin G(Pen G) against the MRSA strains. The most potent of these fiveoxazolidinones, compound 1, produced zones of similar dimensions againstS. aureus to that of penicillin G. Oxazolidinone 2, on the other hand,showed much more moderate activity against both S. aureus and MRSA.Mono-substituted oxazolidinones 3-5 also possessed strong anti-MRSAactivity, surpassing disubstituted derivative 2, indicating thatsubstituents can be placed at either the C₄ or C₅ centers, or at both,without significantly affecting bioactivity. This stands in contrast toprevious observations from studies of mono-versus disubstitutedN-thiolated β-lactams, in which disubstitution on the ring provides forthe best anti-MRSA properties. Additionally, replacement of theN-methylthio moiety of compound 4 for N-sec-butylthio (compound 5) leadsto no significant improvement in anti-MRSA activity.

Enantiomerically paired oxazolidinones 6, 7 and 8, 9 were then evaluatedfor anti-MRSA properties to probe whether absolute stereochemistry was adeterminant of activity (FIG. 3). These four compounds were individuallyprepared from their commercially available N-protio precursors andsubjected to Kirby-Bauer testing. First, from these assays, it was notedthat the phenyl-substituted oxazolidinones 6 and 7 afforded somewhatlarger inhibition zones than the isopropyl-bearing oxazolidinones 8 and9, indicating stronger anti-MRSA activity. Indeed, oxazolidinone 7exhibited a lower broth MIC value (8 μg/mL) against both S. aureus andMRSA than that of oxazolidinone 8 (16 μg/mL). Second, the S enantiomerin each case was found, on average, to be slightly more active than theR-isomer. Indeed, the growth inhibition zones for R-configured compound9 were visibly not as clear as they were for the S-stereoisomer 8,indicative of incomplete growth inhibition. Thus, there may be a smallbut discernible difference in bioactivities of the two enantiomericforms, which should be further evaluated.

Next, the antibacterial capabilities of the oxazolidinones were examinedagainst Bacillus anthracis, the causative agent of anthrax infections,and six other species of Bacillus. Concerns about the possible use of B.anthracis as a biological weapon have led to widespread efforts todevelop antibiotics and vaccines for anthrax infections. For thisinitial examination, N-thiolated 2-oxazolidinones 6-9 were chosen forKirby-Bauer testing. The data shown in Table 2 indicate that each of theN-methylthio 2-oxazolidinones inhibits the growth of all seven speciesof Bacillus. Of these four optically pure compounds, however, 6 and 7had identical activity, while the R compound 9 possessed much weaker andmore sporadic activity compared to that of the S-enantiomer 8. Thereasons for this seemingly anomalous, but reproducible, difference inbioactivity are still under investigation.

TABLE 2 Bacillus species 6 7 8 9 B. anthracis 23 23 23 15 B. globigii 1517 15 17 B. thurigensis 17 15 16 0 B. megaterium 18 20 18 10 B. subtilus19 19 17 19 B. cereus 24 23 22 15 B. coagulans 17 17 17 0

Compound susceptibility measurements obtained from agar disk diffusionof oxazolidinones 6-9 against Bacillus anthracis (Sterne strain) and sixother strains of Bacillus

In each case, 20 μg of the test compound in CH₂Cl₂ was applied to 6 mmcellulose disks prior to inoculation and incubation. The valuecorresponds to average diameter in mm (triplicate experiments) for thezone of growth inhibitions observed after 24 h of incubation at 37° C.Error values of these measurements are ±1 mm.

As previously described for N-thiolated β-lactams, the antibacterialactivity of these agents shows only a small dependence on the ringsubstituents, but requires the N-alkylthio group. In each case,N-thiolated 2-oxazolidinones exhibited antibacterial activity, whereasthe corresponding N-protio oxazolidinones have no antibacterialactivity. It is therefore tentatively postulated that these N-thiolatedoxazolidinones, like their β-lactam counterparts, react covalently withtheir biological target through transfer of the organothio side chain asshown in Reaction 1.

Example 2 Development and Characterization of N-thiolated2-oxazolidinones Procedure for the Synthesis of N-methylthiolated2-oxazolidinones

General Procedure of N-methylthiolation of 2-oazolidinones

A mixture of oxazolidinone (1.0 eq), S-methylphthalimide (1.5 eq) andpotassium carbonate (1.5 eq) in acetone was sonicated till the reactionwas complete. The reaction mixture was diluted with methylene chlorideand filtered through celite. The solvent was removed under vacuum andthe crude product was purified by column chromatography.

4S-(+)-N-methylthio-4-phenyl-2-oxazolidinone (1a)

Isolated 22 mg (34%) as a colorless oil. ¹H NMR (CDCl₃) δ 7.42-7.24 (m,5H); 4.80 (t, J=7 Hz, 1H); 4.62 (t, J=8.8 Hz, 1H); 4.22 (dd, J=7.0, 8.8Hz, 1H); 2.17 (s, 3H). ¹³C NMR (CDCl₃): δ 138.27, 129.82, 129.67,127.91, 70.16, 63.79, 21.20.

4R-(−)—N-methylthio-4-phenyl-2-oxazolidinone (2a)

Isolated 62 mg (48%) as a colorless oil. ¹H NMR (CDCl₃) δ 7.42-7.24 (m,5H); 4.80 (t, J=7 Hz, 1H); 4.62 (t, J=8.8 Hz, 1H); 4.22 (dd, J=7.0, 8.8Hz, 1H); 2.17 (s, 3H). ¹³C NMR (CDCl₃) δ 159.08, 138.28, 129.80, 129.65,127.91, 70.17, 63.75, 21.18.

4S-(−)—N-methylthio-4-isopropyll-2-oxazolidinone (3a)

Isolated as colorless oil. ¹H NMR (CDCl₃) δ 4.24 (t, J=9 Hz, 1H); 4.06(dd, J=9 Hz, 6 Hz, 1H); 3.78-3.71 (m, 1H); d 2.42 (s, 3H); 2.30-2.22 (m,1H); 0.91 (d, J=7.8 Hz, 3H); 0.84 (d, J=6.8 Hz, 3H). ¹³C NMR (CDCl₃)δ159.12, 63.38, 62.32, 28.15, 20.73, 17.53, 14.12

4R-(+)-N-methylthio-4-isopropyl-2-oxazolidinone (4a)

Isolated as colorless oil. ¹H NMR (CDCl₃) δ 4.24 (t, J=9 Hz, 1H); 4.06(dd, J=9 Hz, 6 Hz, 1H); 3.78-3.71 (m, 1H); 2.42 (s, 3H); 2.30-2.22 (m,1H); 0.91 (d, J=7.8 Hz, 3H); 0.84 (d, J=6.8 Hz, 3H). ¹³C NMR (CDCl₃)δ159.12, 63.38, 62.32, 28.15, 20.73, 17.53, 14.12

Additional Studies Detailing Kirby-Bauer Zones of Inhibition ofN-methylthio and N—H Oxazolidinones

TABLE 3

MRSA S. aureus Compound R R′ X (inhibition)^(a) (Inhibition)^(b) 1S-Phenyl H H  0  0 1a S-Phenyl H SMe 29 30 2 R-Phenyl H H  0  0 2aR-Phenyl H SMe 28 29 3 S-iPropyl H H  0  0 3a S-iPropyl H SMe  16^(c) 20^(c) 4 R-iPropyl H H  0  0 4a R-iPropyl H SMe  19^(c)  21^(c)^(a)Reported as median diameter of zone of inhibition, in mm of 10strains of MRSA (652-659, 919-920) ^(b)Median zone of inhibition for S.Aureaus strain 849. ^(c)partial inhibition within this diameter.

The disclosure of all publications cited above are expresslyincorporated herein by reference, each in its entirety, to the sameextent as if each were incorporated by reference individually.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit of the present invention. Accordingly, it isintended that the scope of the present invention be limited solely bythe scope of the following claims.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween. Now that theinvention has been described,

1. A N-thiolated 2-Oxazolidinone compound of the formula:

or a salt or hydrate thereof, wherein: —C(R₁)₃ is aryl, or heteroaryl,alkyl, CH₃, CH(CH₃)CH₂CH₃; —X(R₂)_(n) is hydrogen, methoxy, acetoxy,phenoxy, or X(R₂)_(n) and R₄ forms a spirocycle; R₃ is alkynyl, alkenyl,acetyl, aryl, heteroaryl, aryl(C₁-C₆) alkyl, heteroaryl(C₁-C₆) alkyl,aryl(C₂-C₆) alkenyl, heteroaryl(C₂-C₆)alkenyl, aryl(C₂-C₆) alkynyl orheteroaryl(C₂-C₆)alkynyl; wherein any aryl or heteroaryl is optionallysubstituted with halo, nitro, cyano, hydroxy, trifluoromethyl,trifluoromethoxy, (C₁-C₁₅)alkyl, (C₂-C₁₅)alkenyl, (C₂-C₁₅)alkynyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₁₅)alkoxy,(C₃-C₈)cycloalkyl-(C₂-C₁₅) alkenyl, C₃-C₈)cycloalkyl(C₂-C₁₅)alkynyl,(C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl, (C₁-C₁₅)alkanoyloxy,C(O)O(C₁-C₆)alkyl, C(═O)N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkyl)₂ or H; R₄ ishydrogen, or X(R₂)_(n) and R₄ forms a spirocycle, CH₂N₃, CH₂CH, CO₂Me;and R₅ is hydrogen.
 2. A method of inhibiting a bacterial infectioncomprising administering an effective amount of the N-thiolated2-oxazolidinone of claim 1, to a patient in need thereof.
 3. The methodof claim 2, wherein the bacterium is a Staphylococcus spp.
 4. The methodof claim 2, wherein the Staphylococcus spp. is a methicillin-resistantStaphylococcus.
 5. The method of claim 4, wherein saidmethicillin-resistant Staphylococcus is selected from the groupconsisting of MRSA USF919, MRSA USF920, MRSA USF652, MRSA USF653, MRSAUSF654, MRSA USF655, MRSA USF656, MRSA USF657, MRSA USF658 and MRSAUSF659.
 6. The method of claim 2 wherein the bacterium is a Bacillusspp.
 7. The method of claim 6 wherein the Bacillus spp. is selected fromthe group consisting of B. anthracis, B. globigii, B. thurigensis, B.megaterium, B. subtilis, B. cereus and B. coagulans.
 8. The method ofclaim 2, wherein the N-thiolated 2-oxazolidinone is selected from thegroup consisting of:


9. The method of claim 2 wherein the N-thiolated 2-oxazolidinonecompound is administered in a pharmaceutically acceptable carrier.
 10. Amethod of inhibiting growth of a bacterium comprising the step ofadministering an effective amount of an N-thiolated 2-oxazolidinonecompound, or a salt or hydrate thereof, to the bacterium.
 11. The methodof claim 10 wherein the bacterium is a Staphylococcus spp.
 12. Themethod of claim 2, wherein the Staphylococcus spp. is amethicillin-resistant Staphylococcus.
 13. The method of claim 4, whereinsaid methicillin-resistant Staphylococcus is selected from the groupconsisting of MRSA USF919, MRSA USF920, MRSA USF652, MRSA USF653, MRSAUSF654, MRSA USF655, MRSA USF656, MRSA USF657, MRSA USF658 and MRSAUSF659.
 14. The method of claim 2 wherein the bacterium is a Bacillusspp.
 15. The method of claim 6 wherein the Bacillus spp. is selectedfrom the group consisting of B. anthracis, B. globigii, B. thurigensis,B. megaterium, B. subtilis, B. cereus and B. coagulans.
 16. AnN-thiolated 2-oxazolidinone compound of the formula:

or a salt or hydrate thereof, wherein: —C(R₁)₃ is alkyl, heteroalkyl,alkyl, CH₃, CH(CH₃)CH₂CH₃; —X(R₂)_(n) is OSO₂R₆; R₃ is alkynyl, alkenyl,acetyl, aryl, heteroaryl, aryl(C₁-C₆) alkyl, heteroaryl(C₁-C₆) alkyl,aryl(C₂-C₆) alkenyl, heteroaryl(C₂-C₆)alkenyl, aryl(C₂-C₆) alkynyl orheteroaryl(C₂-C₆)alkynyl; wherein any aryl or heteroaryl is optionallysubstituted with halo, nitro, cyano, hydroxy, trifluoromethyl,trifluoromethoxy, (C₁-C₁₅)alkyl, (C₂-C₁₅)alkenyl, (C₂-C₁₅)alkynyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyl(C₁-C₁₅)alkoxy,(C₃-C₈)cycloalkyy-(C₂-C₁₅) alkenyl, C₃-C₈)cycloalkyl(C₂-C₁₅)alkynyl,(C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl, (C₁-C₁₅)alkanoyloxy,C(O)O(C₁-C₆)alkyl, C(═O)N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkyl)₂, or H; R₄ ishydrogen, CH₂N₃, CH₂CH, CO₂Me; R₅ is hydrogen; and R₆ is alkyl, aryl, orheteroaryl, wherein any aryl or heteroaryl is optionally substitutedwith halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy,(C₁-C₁₅)alkyl, (C₂-C₁₅)alkenyl, (C₂-C₁₅)alkynyl, (C₃-C₈)cycloalkyl,(C₃-C₈)cycloalkyl(C₂-C₁₅)alkoxy, (C₃-C₈)cycloalkyl-(C₂-C₁₅)alkenyl,(C₃-C₈)cycloalkyl(C₂₁-C₁₅)alkynyl, (C₁-C₁₅)alkoxy, (C₁-C₁₅)alkanoyl,(C₁-C₁₅)alkanoyloxy, C(O)O(C₁-C₆)alkyl, C(═O)N((C₁-C₆)alkyl)₂, orN((C₁-C₆)alkyl)₂.